Development, qualification, validation and application of the neutral red uptake assay in Chinese Hamster Ovary (CHO) cells using a VITROCELL® VC10® smoke exposure system. (April 2017)

• Record Type: Journal Article
• Authors: Fields, Wanda; Fowler, Kathy; Hargreaves, Victoria; Reeve, Lesley; Bombick, Betsy
• Is Part Of: Toxicology in vitro. Volume 40(2017)
• Page Start: 144
• Page End: 152
• Publication Date: 2017-04
• Subjects: Smoke exposure -- Neutral red uptake -- CHO cells -- Eclipse -- VITROCELL® -- ALI; Toxicity testing -- In vitro -- Periodicals; Toxicology -- Periodicals; 615.9
• DOI: 10.1016/j.tiv.2017.01.001
• Languages: English
• ISSNs: 0887-2333

Abstract

Abstract: Cytotoxicity assessment of combustible tobacco products by neutral red uptake (NRU) has historically used total particulate matter (TPM) or solvent captured gas vapor phase (GVP), rather than fresh whole smoke. Here, the development, validation and application of the NRU assay in Chinese Hamster Ovary (CHO) cells, following exposure to fresh whole smoke generated with the VITROCELL® VC10® system is described. Whole smoke exposure is particularly important as both particulate and vapor phases of tobacco smoke show cytotoxicity in vitro. The VITROCELL® VC10® system provides exposure at the air liquid interface (ALI) to mimic in vivo conditions for assessing the toxicological impact of smoke in vitro. Instrument and assay validations are crucial for comparative analyses. Goals of this study: 1) demonstrate functionality of the VITROCELL® VC10® system by installation, operational and performance qualification, 2) develop and validate a cellular system for assessing cytotoxicity following whole smoke exposure and 3) assess the whole smoke NRU assay sensitivity for statistical differentiation between a reference combustible cigarette (3R4F) and a primarily "heat-not-burn" cigarette (Eclipse). Results: The VITROCELL® VC10® provided consistent generation and delivery of whole smoke; exposure-related changes in in vitro cytotoxicity were observed with reproducible IC50 values; comparative analysis showed that the heat-not-burn cigarette was significantly (P < 0.001) less cytotoxic than the 3R4F combustible cigarette, consistent with the lower levels of chemical constituents liberated by primarily-heating the cigarette versus burning. Highlights: Data from the development and validation of an assay for assessing cytotoxicity of smoke at ALI are presented. The VITROCELL® VC10® system was validated by equipment qualification protocols. Differentiation between cytotoxicity of 3R4F and Eclipse cigarettes was demonstrated.